Mechanisms Involved in Degradation of Human Insulin by Cytosolic Fractions of Human, Monkey, and Rat Liver

The degradation of native and ¹²⁵I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH₂-terminal sequence analysis to be intact A chain, B chain, and des(Phe¹)–B chain. Human liver cytosol also formed ¹²⁵I–A chain and ¹²⁵I–B chain as major products when specifically labeled ¹²⁵I–HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn²⁺. In contrast, monkey and rat liver cytosol proteolytically degraded ¹²⁵I–HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn²⁺-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA. When proteolysis was inhibited in monkey cytosol, the formation of B chain, reflecting the activity of PDI, was evident. Immunoinhibition of PDI with ascites containing monoclonal antibodies to PDI prevented the metabolism of ¹²⁵I-(B26)HI in monkey and Mn²⁺ supplemented human liver cytosol. PDI antibodies did not inhibit the proteolysis of ¹²⁵I-(B26)-HI in rat cytosol or the proteolysis of ¹²⁵I-(B26)–B chain by monkey or Mn²+-supplemented human liver cytosol. These results are consistent with the involvement of both PDI and IDE in the degradation of HI by nonrodent and human liver cytosol.